/. Embryol exp. Morph. Vol. 27, 2, pp. 317-337, 1972
Printed in Great Britain
317
Cell movements in morphogenesis of
hydroid polypes
By L. V. BELOUSSOV, 1 L. A. BADENKO, 2
A. L. KATCHURIN 2 AND L. F. KUR1LO (FILATCHEVA) 1
From the Department of Embryology, Moscow University,
and the Physico-Technical Institute, Leningrad
SUMMARY
1. Growth, morphogenesis and cell movements were studied in Obelia loveni, O. geniculata
and Dynamena pumila with the use of time-lapse cinematography, visual observations of
vitally stained objects and by histological techniques.
2. Growth pulsations with the period around 14min and the amplitude around 15 ju,m
exist in Dynamena pumila and with the period 5-8 min and amplitude up to 5 /*m in
Obelia loveni. It was demonstrated that the rhythm of growth pulsations does not coincide
with the rhythm of periodical contractions of the proximal part of coenosarc.
3. The distalwards movements of individual cells in the ectoderm of growing stems and
hydranth rudiments are described. A considerable variability in the rates of movements of
ectodermal cells has been demonstrated.
4. Different kinds of cell reorientations in developing rudiments are described. As a rule,
they precede the alterations of growth directions or of rudiment shapes.
5. The mechanisms involved in deformations of epithelial layers are discussed.
6. The possibility of the existence of passive, elastico-plastic structures in the deforming
epithelial sheets is suggested.
INTRODUCTION
The problem of co-ordination of cell movements during morphogenesis
attracts special attention nowadays. It has been extensively studied in sea-urchin
embryos (Gustafson & Wolpert, 1967) and several other species. Recently a
number of investigators have concluded that hydroid polypes also are suitable
organisms in this respect. The simplicity and geometrical regularity of their
shape and the relative uniformity of their cellular structure permits the hope
of working out some general principles of the organic formation of shape.
A comprehensive review of Hydrozoa morphogenesis was made by Webster
(1971). Many (perhaps, a majority of) studies mentioned there deal with the
problems concerning 'global' morphogenetic patterns - that is, determination
of polarity, general subdivision of a body, etc. More 'local' problems concerning
the morphogenetic activities of individual cells and their spatial and temporal
1
Authors' address: Department of Embryology, Moscow University, Moscow 117234,
USSR.
2
Authors' address: Physico-Technical Institute, Academy of Sciences USSR, Politechnicheskaia ul. 26, Leningrad K-21, USSR.
318
L. V. BELOUSSOV AND OTHERS
co-ordination seem as yet to be insufficiently studied, especially in marine
Hydrozoa. The task of this paper is to describe these aspects of morphogenesis
in some marine Hydrozoa, belonging to subclass Thecaphora.
An important role of cell movements in establishing morphological patterns
of Hydrozoa has been stressed by various authors (for review of data on marine
Hydrozoa see Berrill, 1961). According to a number of authors (Beloussov,
\96la, 1963;Hale, 1960,1964;Crowell,Wyttenbach&Suddith,1965;Filatcheva,
1966; Campbell, 1967) cell proliferation does not play any direct role in
determining the shape or even the dimensions of rudiments. As to the temporal
pattern of cell movement, some years ago an interesting phenomenon of growth
pulsations in the tips of Hydrozoa outgrowths was described (Beloussov, 1961 b;
Hale, 1964; Wyttenbach, 1968). The most detailed description of the phenomenon was made on Campanularia stolons by Wyttenbach. The following points
of his work should be mentioned here: growth pulsations of a stolon tip originate
independently from the periodical contractions of the proximal part of the
coenosarc; the duration of the pulse cycle is about 6 min at 20 °C and decreases
along with the rise of temperature. At constant temperature, cycle duration is
much more stable than the amplitude of pulsations. A periodical thinning of the
ectodermal layer correlated with the upward shifts of the entodermal layer was
also observed.
These conclusions will be later compared with the original data. In the present
paper the results of investigations of growth pulsations and of the morphogenetic
movements of individual cells are described and some problems concerning the
mechanisms of deformations of epithelial sheets are discussed.
MATERIALS AND METHODS
Three species of marine Hydrozoa (Thecaphora) have been studied: Obelia
loveni, O. geniculata and Dynamena pumila. The samples were collected on the
tide lands of the White Sea. The present paper deals only with the development
of the vegetal generation.
The following methods of vital observations were employed:
(1) Time-lapse films. Several periods of stem growth and hydranth formation
in Obelia loveni and Dynamena pumila were registered on 35 mm film. Exposure
intervals ranged from 0-5 min to 2 min. The duration of continuous filming
was up to 12 h. A 'Convas' camera was used, combined with microscope
MBI-3. The microscope objectives were x 3 and water immersion x 40. The
samples were immersed in a 20 ml glass dish, being attached to its bottom by an
adhesive tape. The temperature varied from 15 to 20 °C.
(2) Vital staining. Stems and hydranths of Obelia loveni were immersed for
40-60 min in weak solutions of Nile blue sulphate. Then they were transferred
to pure sea water and fixed horizontally; 1-2 h later the staining became granular.
The granules were localized in ectodermal cells exclusively. The movement of
Morphogenesis of hydroid polypes
319
several (up to four) granules was continuously traced during 2-3 h with the use
of a water-immersion objective, x 40.
3. For histological purposes samples of colonies were fixed in a Bouin solution and embedded in paraffin wax or paraffin-celloidin; 8-12 /mi sections
were prepared. The slides were stained with Heidenhain's iron haematoxylin.
RESULTS
I. General description of structure and development of
vegetal generation of the studied species
A. Some notes on histological structure
The body of a Hydrozoa colony (usually called its coenosarc) consists of two
cell layers (ecto- and entoderm), each being mainly composed by myoepithelial
cells. There are some important differences in the structure of ecto- and entodermal myoepithelial cells. Whereas the entodermal cells are more epitheliallike and are bound together in a single sheet, the ectodermal ones look like the
bundles of fine contractile tonofibrils. They are not strictly connected, but are
firmly attached to the common surface membrane, enveloping the ectodermal
layer. The surface membrane is not to be confused with the chitinous hydrotheca, or perisarc, which is secreted by special glandular ectodermal cells and
which is laid down at the external side of the surface membrane. If a part of
coenosarc contracts (e.g. when the hydranth 'neck' is formed, see below), its
perisarc remains fixed and may therefore be used as a stable point of reference.
B. The external character of branching and hydranth development
Obelia loveni and O. geniculata (Fig. 1). The branching of the colonies is
sympodial.1 The branches of both species are bent, in O. geniculata the bending
being more pronounced than in O. loveni. The new stems are initiated either just
below the growing tip of the maternal stems or on the proximal parts of the
colony. In the first case the new (daughter) stems are always formed at the convex
side of the maternal ones (Fig. 1 A, d.s.) and grow in the same plane. Just after
their initiation the daughter stems grow parallel to the maternal ones (Fig. 1B,
d.s.), but later their tips deviate sideways (Fig. 1C, D, d.s.). In the proximal
regions new stems are initiated in the axiles of the old ones. The plane of
bending of the first ones is in no way correlated with the plane of bending of the
latter.
As stem growth proceeds, several circular grooves are formed one after
another at some distance from the tip (Fig. 1B, C, grx), fixed by the chitinous
perisarc. In general, about 3-4 such grooves are formed, and then a period of
'smooth' growth takes place, being in its turn succeeded by a new period of
1
This type of branching is characterized by a replacement of an old growth point by a
new one after the formation of each new outgrowth; every growth point arises proximally to
the old one.
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L. V. BELOUSSOV AND OTHERS
D
Fig. 1. Successive stages of development of stem and hydranth in Obelia loveni
(pictures from time-lapse film), d.s., Daughter stem; grx, first series of grooves;
gr2, second series of grooves; h.r., hydranth rudiment; n, hydranth 'neck'; dp,
diaphragm; a, side of a hydranth, adjacent to maternal stem; b, side away from
maternal stem; ps, perisarc.
groove formation (Fig. 1D-G, gr2). Later the part of the stem located distally
from the last groove expands and elongates, thus forming a rudiment of a
hydranth (Fig. 1E, h.r.). The proximal part of the rudiment is constricted, forming a hydranth's 'neck' (Fig. IF, G, n). A thin cellular plate, a diaphragm, develops proximally to the neck (Fig. 1G, dp). The most expanded part of the rudiment, situated distally to its neck, later splits into a series of columnar tentacle
rudiments, whereas its roof transforms to a hypostome. A hydranth becomes
slightly asymmetrical in the plane of the stem bending: the side of a hydranth
adjacent to the maternal stem (Fig. 1E-G, a) is slightly more convex than the
opposite side. At the same time the hydranth is completely symmetrical in the
plane perpendicular to that of stem bending.
Dynamena pumila (Fig. 2). The branching is monopodial.1 The vertical
branches are composed of a series of storeys, each including a pair of identical
lateral (LR) and a central rudiment (CR) - Fig. 2 A. The U?'s are bent distally
(Fig. 2B-D) and later transform to hydranths, whereas the CR remains
undifferentiated and grows farther. At first its tip is narrowed, but later it
expands and is transformed to a spherical (Fig. 2B) and then to a triangular
(Fig. 2D) rudiment. The plane of its expansion completely coincides with that
of preceding CR. Thus the whole colony is flattened in a single plane (Fig. 2B1?
D l5 Ex). After expansion each new CR is split by two vertical furrows into three
rudiments (Fig. 2E l5 Li?1, CR1), the lateral ones being again transformed to
hydranths, whereas the central one grows farther. Similar to Obelia hydranths,
the ZJ?'s are subdivided into a diaphragm, a constricted proximal part and an
expanded distal part (Fig. 2D, dp, n).
1
This type of branching is characterized by a prolonged action of a single growth point
which is situated at the distal pole of the growing stem.
Morphogenesis of hydroid polypes
321
100//m
A
LR
Fig. 2. Successive stages of development of growing tip in Dynamena pumila
(pictures from time-lapse film). A-D, Side view; Br-Ex, top view. CR, Central
rudiment; LR, lateral rudiment. B, C, The opposite phases (extension and contraction) of the same growth pulse, (dp, Diaphragm; n, hydranth neck.)
The following general features of the described morphogenetic processes are
to be emphasized: in respect to the directions of their growth, the abovementioned rudiments are, as a rule, either asymmetrical (Obelia) or anisotropic
(Dynamena). The plane of anisotropy or asymmetry remains constant for a
considerable part of colony.
II. The character of growth of Obelia loveni and Dynamena pumila
according to time-lapse films
By means of time-lapse cinematography the pulsatory character of the
growth, the relations between growth pulsations and coenosarc contractions
and between the rates of migration of the tip cells of both layers (ecto- and entoderm) have been revealed.
A. Growth pulsations
The pulsatory character of growth is most obvious in CR of Dynamena pumila
(Fig.. 2B, C; Fig. 3). Short (2-4 min) periods of rudiment contractions
(Fig. 2C) are alternated with the more prolonged (10-13 min) of its extension
(Fig. 2B). Thus the duration of the whole cycle is about 12-15 min, its amplitude
about 15-20 jtim. The average rate of growth is fairly constant during the whole
period of observations. No obvious correspondence has been observed between
the phases of tip pulsations and coenosarc contractions (the latter measured by
the length of the coenosarc diameter along B-B level, Fig. 3). This corresponds
to the results obtained on Campanularia stolons (Wyttenbach, 1968).
Similar growth pulses without obvious correlations with coenosarc contractions are observed on LR's. However, due to the asymmetry of the rudiments,
it is difficult to represent these results graphically.
0
Time (min)
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
0
5
10
A
15 20
25 30
0
10 15 20 25
30 35 40 45
Time (min)
50
0
5
10 15 20 25
30 35 40 45 50 55
Fig. 4. Pulsations of growing tip in Obelia loveni. A, B, C, Successive periods of stem growth, separated by 1 h periods.
Abscissa: time of observation. Ordinate: position of a growing tip.
10
15
20
Fig. 3. Pulsations of growing tip (a) and coenosarc (b) in Dynamena pumila. 1, Initial; 2, final stage of a studied period.
B-B indicates the level of the measured coenosarc diameter. The dotted arch at 2 indicates the position of the distal surface of
the rudiment at the initial stage 1. Abscissa: time of observation. Ordinate: position of a growing tip (a) and diameter of
coenosarc (b).
300
270
240
210
180
150
120
90
60
30
PI
O
H
O
GO
a
C/5
o
a
to
Morphogenesis of hydroid polypes
/
/lO
323
40 min
5 10 15 20 25 30 35 40 45 50 55 60 65
Time (min)
Fig. 5. Growth pulsations and fluctuations of shape in Obelia loveni hydranth.
A, Fluctuations of shape of right side wall of a hydranth; • , contours with most
relief. B, The contours of the whole rudiment, corresponding to 1 (solid line) and
2 (broken line) in A. C, Correlation between the fluctuations of a growing tip (1)
and contractions of coenosarc (2); Abscissa: time of observation. Ordinate:
position of a tip (1) and diameter of coenosarc (2).
In Obelia loveni the amplitude of tip pulsations is smaller and less constant.
However, using water-immersion x 40, periods of 5 min pulsations are constantly observed in Obelia stems and those of 8 min in Obelia hydranths. The
period of pulsation is much more stable than its amplitude (Fig. 4).
In developing Obelia hydranths the periodical shape alterations correlated
with growth oscillations can be observed. As a rule, in the phase of maximal
contraction the outlines of a hydranth roof become smooth, the protuberances
of the tentacles and hypostome disappear. On the contrary, during the extension
phase the rudiment outlines become more pronounced and the mentioned protuberances become visible again (Fig. 5A, B).
In Obelia stems as well as in Dynamena the rhythm of growth pulsations does
not coincide with the rhythm of coenosarc contractions (Fig. 6B). In hydranth
rudiments, however, both rhythms seem almost completely to coincide (Fig. 5C).
The hydranth rudiments behave thus as a unitary contractile system.
B. Correlations of migration rates of ecto- and entodermal tip cells in Obelia
outgrowths
According to time-lapse data, three different kinds of rate correlations are
taking place:
324
L. V. BELOUSSOV AND OTHERS
/mi
120
100
80
60
40
20
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0
5
10
15 20
25
30
35
40
45
50
55
cct.
.-•'•• end.
60
Time (min)
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200
180 160 140 - •• "'"
120
100
80 A A '
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20 BB
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I
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1
0
10
20
30
40
50
60
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80
1
1
Ji
90 100
Time (min)
/mi
200
180
160
140
120
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80
60
40
20
-
....• ect.
I
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-
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0
40
120 160 200 240
Time (min)
Fig. 6. Different kinds of rate correlations of ecto- and entodermal tip cells in Obelia
loveni. AA, BB, contractions of coenosarc diameters at the levels indicated at right.
Abscissae: time of observations. Ordinates: positions of the tips of ectodermal (ect.)
and entodermal (end.) layers and diameter of coenosarc (AA, BB).
(1) The rates of movement of tip cells in both layers are approximately equal
(Fig. 6 A). This situation is mainly typical of the smooth growth period when
no grooves appear (a period between the formation of n± and n2, Fig. 1D).
(2) The rate of movement of ectodermal tip cells exceeds that of the entodermal cells. As a result, the thickness of the ectodermal layer can be approximately doubled (Fig. 6B). This situation is mainly typical of the first period of
groove formation (just after the initiation of a stem) - Fig. 1B, nx.
(3) The rate of migration of ectodermal cells is less than that of entodermal
cells (Fig. 6C). As a result, the thinning of the ectodermal layer and the more
compact arrangement of the tip cells of the two layers take place. The situation
is typical of the early stages of hydranth development. Later the reorientation
Morphogenesis of hydroid polypes
325
of cells of both layers and the expansion of the rudiment take place (see details
below).
The described kinds of rate correlations are much more various than those
described by Wyttenbach for Campanularia stolons. Wyttenbach observed
only a periodical 'pushing' of ectodermal cells by entodermal ones, which led
him to conclude the passivity of ectoderm. Meanwhile, in vertical Obelia stems
along with the analogous rate correlations (type 3), quite the opposite (type 2)
is observed. The latter is incompatible with the assumption of passive ectodermal
shift. As a whole, the existence of the different types of rate correlations demonstrates the autonomous migratory activity of cells of each layer.
III. The action of metabolic inhibitors on the shape of the
hydranth rudiment in Obelia loveni
Pieces of Obelia colonies, including hydranth rudiments, were immersed in
0-05 M-KCN or 1-2 % dinitrophenol at different times after the beginning of
contraction of its proximal part (i.e. neck formation, Fig. 1F). In samples immersed not later than 1 h after the beginning of neck formation, the latter completely disappeared in ^ h; in other words, the proximal parts of the rudiments
expanded again (23 cases out of 26). Vital staining and histological examination
did not reveal any signs of cell degeneration at the time of expansion. In samples
immersed in inhibitor solutions later than 1 h after neck formation, the latter
failed to expand even if complete cell degeneration took place. Thus, one may
assume the existence of a certain critical point in hydranth morphogenesis after
which the contraction of the proximal part is stabilized.
IV. Morphogenetic movements of individual cells
A. Distalwards movements of ectodermal cells according to observations in vivo
Observing O. loveni outgrowths, stained in vivo, the movements of the individual ectodermal cells were traced. The main results of the observations were
as follows.
The cells situated at the distal region of growing stem and hydranth rudiment
migrate distalwards at a rate approximately equal to the growth rate of the whole
stem (around 30 /*m/h). At the same time a considerable disparity in migratory
rates of individual cells was revealed (Fig. 7). In order to evaluate the degree of
rates dispersion and its dependence on the relative positions of the observed
cells, a number of proximal (situated between two neighbouring grooves) and of
distant (separated by grooves or situated at the opposite sides of the stem) cells
were traced. In all, the movement of 48 pairs of proximal stem cells, 24 pairs of
distant stem cells, 35 pairs of proximal hydranth cells, 35 pairs of analogous
proximal cells of hydranth rudiment and 36 pairs of distant hydranth rudiment cells was traced and the differences of simultaneous 10 min distalwards
cell tracks were measured. The cumulative cell rate differences distribution
21
E M B 27
326
L. V. BELOUSSOV AND OTHERS
10 20 30 40 50 60
Time (min)
Fig. 7. Trajectories of migration of four ectodermal cells in O. loveni stem. Left,
graphics; right, cell arrangement in growing stem. Abscissa: time of observation;
ordinates: positions of observed cells.
curves were plotted and were compared with the corresponding theoretical
curves of normal (Gauss) distribution (Fig. 8). One can see that the dispersion
in cell rates for the proximal cells does not differ considerably from that for distant cells. The rate differences distribution curves for stem cells were distinctly subnormal (excess index e = 2-2 + 032), whereas the similar curves for
hydranth rudiment cells did not differ sufficiently from the corresponding Gauss
curve (e = 0-24 ±0-36). These results demonstrate that the distalwards migration of stem cells is to some extent more co-ordinated than that of hydranth
cells. However, in both cases the difference in rates of even proximal cells is
great enough to demonstrate the lack of strict connexions between them. That
seems to be the most important conclusion from the above data.
At the more proximal stem regions the cell movement is of a pulsatory character, without a significant distalwards shift. Here the cell shifts seem to be
correlated with coenosarc contractions and may be regarded as passive.
B. Cell movements at the growing stem tip according to histological data
The growth of stem tip consists of sliding distal shifts of the central group of
cells. In some cases these shifts are relatively rapid (about 90 /tm/h) and spraylike (Fig. 9 A). As a rule, however, the rate of the distal shifts of tip cells does not
exceed 30 /tm/h. The successive phases of the shift are presented on Fig. 9B-D.
Morphogenesis of hydroid polypes
327
100 -
90
80
70
60
50
40
20
10
A//m (10 min)
Fig. 8. Cell rates differences distribution curves (cumulates). Abscissa: differences
in rates of simultaneous migration of cell pairs (/tmper 10 min). Ordinate: percentage
of cell pairs. Solid lines, stem cells; dotted lines, hydranth cells. O, Distant cells;
x, proximal cells; A, theoretical curves of normal (Gauss) distribution. Right
below, a scheme of arrangement of pairs of proximal (ab), and distant (ac, be, cd, bd,
ad) cells.
One can see that after the extension of the ectodermal cells the rupture of basal
membrane takes place (Fig. 9, r.b.m.), obviously due to the pressure of entodermal cells, which later also shift distalwards.
The sliding shift of the central cell group in respect to lateral ones as well as
the alternating character of ecto- and entodermal cell shifts demonstrate the
lack of close connexions between the tip cells inside each layer and between
both layers.
C. Reorientations and deformations of cells
One of the most interesting features of cell behaviour in Hydrozoa morphogenesis seems to be various reorientations and deformations of cells, regularly
correlated with the shape alterations of the whole rudiment and as a rule
preceding the latter.
328
L. V. BELOUSSOV AND OTHERS
r.b.m.
b.m.
D
Fig. 9. Different phases of cell shifts in the growing tip of Obelia stem.
b.m., Basal membrane; r.b.m., rupture of basal membrane.
(a) Reorientations in bending rudiments. The orientation of cells in straightly
growing rudiments is symmetrical. Often their arrangement at the rudiment
tips is similar to that of the onion scales (Fig. 9D). However, in the rudiments
which are to be bent soon, cell orientation becomes asymmetrical. In such rudiments the bisector of the angle formed by the axes of cells at the opposite walls
deviates considerably from the rudiment axis in the direction of the presumptive
bending (Fig. 10A, B). The corresponding angle varied from 6° to 25° (17° on
Morphogenesis of hydroid polypes
329
K
B
Fig. 10. Cell orientation in stems of Obelia geniculata (A) and O. loveni (B) just
prior to their bending. AO and BO indicate cell orientation in the opposite stem
walls. OK, Bisector of angle AOB. Dotted lines indicate median axes of stems.
average) for Obelia loveni (6 measurements) and from 9° to 30° for O. geniculata
(20° on average, 5 measurements). No cases of negative or zero-deviation
occurred.
The correlation between cell orientation and the direction of rudiment
growth was demonstrated experimentally in D. pumila (for details see Beloussov,
1965). Normally the cell arrangement in the LR's of D. pumila is clearly asymmetrical, correlated with the presumptive bending of these rudiments. However,
if we remove CR, the cell arrangement in the LR's becomes symmetrical, which
corresponds with the vertical direction of their succeeding growth. In CR's the
situation is the opposite. Normally it grows straightly vertical and the cells are
arranged symmetrically in its walls (Fig. 11 A). After the removal of Li?, however, the cells of the wall, adjacent to the removed rudiment, become more perpendicular to the long axis of the colony. As a result, the whole cell arrangement
becomes asymmetrical (Fig. 11B). Such CR's later grow asymmetrically, bending
towards the removed LR. In all cases described above, either normal or experimental, cell reorientation regularly precedes the bending of the whole rudiment.
(b) Reorientations and deformations of cells during the development ofhydranth
of Obelia loveni and CR of Dynamena pumila. Before the beginning of the expansion of the growing tips in Obelia and Dynamena the orientation of their
330
L. V. BELOUSSOV AND OTHERS
CR
B
Fig. 11. Cell orientation in side walls of CR in Dynamena pumila.
A, Normal case; B, after the removal of the right LR.
cells only slightly deviates from the vertical. The lateral cell poles are always
situated more distally than the median ones. At the early stages of hydranth
(resp. CR) formation the cells of both their layers begin to rotate, tending to
orient transversely (Fig. 12). This rotation is especially pronounced in the distolateral parts of the rudiments, which are mostly expanded. At the advanced
stages the most complicated cell reorientations take place in the ectoderm
(Fig. 13). At the intermediate stages the cells become U-shaped (Figs. 13B,
14 A) and in some cases S-shaped (Fig. 13C). Later they become oriented proximally by their lateral ends (Fig. 13E). Therefore, the whole angle of their rotation
is about 90° (compare Fig. 13 A and E). The part of the rudiment composed of
such reversed cells later contracts and transforms to its neck. Meanwhile cell
orientation in more proximal regions (near the rudiment bases) remains unchanged. As a result, a narrow zone of counter-oriented cells is established near
the hydranth (resp. CR) base (Fig. 13E, dp). Later it transforms to a diaphragm.
One can see from Fig. 13 that during cell orientations the wall of a hydranth
rudiment does not considerably alter its shape. At the same time cell reorientations are closely correlated with the presumptive deformations of the sheet.
It can be demonstrated geometrically: if we prolong the cell axes (in the case of
D. pumila CR, the distal parts of the axes) and plot a curve, perpendicular to all
331
Morphogenesis of hydroid polypes
90
80
J70
,; 60
•_
o
50
•|
40
| 30
O
20
10
50
100 f
150
Length of hydranth rudiment, L
200
Fig. 12. A diagram of cell reorientation at the early stages of development of
O. loveni hydranth (left below, initial stage; upper right, final stage of a given
period). The arrow on the abscissa indicates the beginning of transverse extension
of a rudiment, - x - , Ectodermal cells; —O—, entodermal cells.
\
A
B
C
Fig. 13. Reorientations of ectodermal cells in left side wall of O. loveni hydranth
at successive stages of its development, dp, Rudiment of diaphragm.
332
L. Y. BELOUSSOV AND OTHERS
Fig. 14. Three successive stages of development of CR in Dynamena pumila.
The line perpendicular to the distal segments of the ectodermal cell axes is
approximately similar to the contour of the succeeded stage.
these segments, the curve is obviously similar to the outline of the succeeding
stage of the given rudiment (Fig. 14).
Specific alterations of cell shape and arrangement take place in the ectoderm
of the hydranth rudiment roof in Obelia. The early steps of hydranth formation
are characterized by the extensive shortening of the roof ectodermal cells (compare Fig. 9B-D and Fig. 15A, B). Somewhat later they become bow-shaped
Morphogenesis of hydroid polypes
333
Fig. 15. A,B»Cell arrangement in the roof of a rudiment of 6). /ovemhydranthattwo
successive stages of its development. Bl5 A schematic representation of the contours
of hydranth roof and of the arrangement of cell nuclei at stage B.
(Fig. 15A), then they straighten out again (Fig. 15B). At this stage the cell
arrangement in the rudiment roof is in many cases more relief than the outlines
of the rudiment surface (Fig. 15Bi; compare the dotted line which is parallel
to the arrangement of cell nuclei with the solid line which is parallel to the rudiment surface). One can see that the line of nuclei arrangement is similar to the
dotted line on Fig. 5B, while the outline of the hydranth surface is similar to the
334
L. V. BELOUSSOV AND OTHERS
solid line of the same figure. Remembering that the two lines reflect different
phases of shape pulsations, one may suppose that during the phase of smoothing
of the external surface there are individual cells (or their nuclei) which retain the
pattern of the 'relief phase, i.e. the pattern of the morphodifferentiation of a
hydranth. Therefore the individual cells may be regarded as the active elements
of the rudiment, whereas the common surface membrane of a layer seems to be
more passive.
DISCUSSION
The phenomena described above pose several problems, some of them being
narrower, whereas others are more general. The discussion aims rather to outline these problems than to suggest solutions.
1. Pulsatory character of stem and hydranth growth
This phenomenon undoubtedly requires detailed study. Several aspects seem
to be of special interest here. First of all, the existence of such pulsations demonstrates a certain synchrony in behaviour (that is, in degree of extension-contraction) of a large group of tip cells. The origin of this synchronization remains
completely unknown. A narrower problem is the relation between tip pulsations
and the contractions of the proximal regions of coenosarc. Our data reveal no
direct correlation between the two, but perhaps some sort of indirect interdependence between these processes nevertheless exists.
The biological role of growth pulsations seems to be clearer: it is the way to
ensure a simultaneous pressure of the whole group of tip cells on the passive
sheet membranes and thus to deform or rupture the latter. Without synchronization of cell shifts the mechanical resistance of the membranes (first of all, the
basal membrane) could not be overcome.
It is to be stressed, however, that cell shifts are synchronous only at the rudiment tips, whereas, according to in vivo observations, the migration of the more
proximally situated cells demonstrates a considerable dispersion of rates at every
given period.
2. Cell reorientations and mechanisms of deformation of epithelial layers
Our data suggesting an important role of cell reorientations in Hydrozoa
morphogenesis are in agreement with those obtained on freshwater Hydra and
several other species (Webster, 1971). This author attributes to cell reorientations
a leading role in determining the direction of morphogenetic gradients. This interesting problem is, however, beyond the scope of the present paper. The only
aspect to be discussed here is the information about the mechanisms of epithelial
deformations which may be derived from this phenomenon.
The problem of the nature and distribution of forces which deform a cell
layer in normal development nowadays attracts the attention of several investigators. Some of them suppose that the changes in epithelial shape are
Morphogenesis of hydroid polypes
335
caused by active expansion or contraction of its surface (Baker & Schroeder,
1967). Another point of view is that the deformation of a sheet is an immediate
result of shape alterations of the cells, both processes occurring simultaneously
(Gustafson & Wolpert, 1967). For example, the imagination of sea-urchin
entoderm these authors regard as a result of spherulization of its cells, leading
to the extension of the whole rudiment surface. Changes in the adhesive cell
properties are regarded as a cause of alterations of cell shape.
Therefore, according to the first conception, the surface of a sheet behaves
like an active structure, while according to the latter one the active role is
played by the individual cells. At the same time both conceptions coincide in
assuming the simultaneity of the deformations of the individual sheet cells and
the sheet as a whole.
In Hydrozoa, however, we see a different picture. As was mentioned above,
the regular reorientations of the individual cells take place considerably earlier
than the sheet alters its shape. Thus, the deformation of the epithelial sheet consists of two phases: (1) the phase of cell reorientation without a significant
deformation of the whole sheet (latent phase); (2) the phase of a visible deformation of the sheet due to the shifts of already reoriented cells parallel to their
axes. It may well be that two similar phases occur also in a number of other
morphogeneses; for example, the convergence of the dorsal poles of the neural
plate cells takes place considerably earlier than the plate starts to evaginate. In
this case the problem of epithelial deformation may be separated into two
main questions: (a) What is the cause of cell reorientation ? (b) What are the
mechanisms of cell shifts, succeeding cell reorientations ?
(a) This problem may be discussed now only in general terms. So far as cell
reorientations determine the regular morphological pattern of the entire rudiment, one must seek for the explanation of the regular spatial distribution of the
reorienting forces. This kind of problem belongs to the realm of morphogenetic fields theories, or to its newest modification, a 'positional information'
concept (Wolpert, 1969). On the other hand, observing cell reorientations (e.g.
on Fig. 13), one can see that they need a number of separate forces of various
magnitudes and directions, applied to individual cells and even to different parts
of a cell. It is hard to imagine such local forces to be originated from a single
source, removed from the reorienting cells. On the other hand, it seems to be
natural to assume the origin of the reorienting moment in the immediate
neighbourhood of a given cell. Therefore one may suppose that the 'positional
information' which determines cell reorientation is transmitted to each cell by
the neighbouring cells. Some years ago a similar model was proposed by one of
us (Beloussov, 1968). In this model cell reorientations were regarded as a result
of the asymmetrical position of a given cell in respect to the adjacent cells. For
example, if a certain cell was surrounded by two others, one of the latter (A)
forming a larger angle with the axis of the central cell than the other (B), the
first cell tended to rotate towards A-cell. This model gave a formal explanation
336
L. V. BELOUSSOV AND OTHERS
to cell reorientations described above. The nature of the forces involved was
not, however, discussed. Further investigations are needed here.
(6) One can see that during the 'post-reorienting' cell shifts the shapes of the
individual cells are transformed from non-symmetrical (parallelogram-like or
bent) to more symmetrical, often rectangular (Fig. 14, compare B and C). If we
assume that each cell possesses an intrinsic tendency for symmetrization and at
the same time can serve as a support for the shifts of the neighbour cells, the
specific pattern of deformation of the whole sheet can be derived. The symmetrization tendency of the individual cells seems to be, in its turn, a natural
phenomenon. One can interpret it, for example, as a result of the tendency of the
energy of the cell surface to decrease. Undoubtedly, however, this explanation is
purely speculative and the problem requires further study.
3. Deforming-resistant structures in epithelial sheet
The possibility of reversing the contraction of the proximal part of the
hydranth rudiment by means of metabolic inhibitors may be interpreted as a
result of the existence of elastic deforming-resistant structures, which under
normal metabolic conditions are overcome by shifting cells, but reveal their
tendency to contract after cell depression. If, however, the duration of sheet
deformation is sufficiently long, these structures lose their elastic properties
and behave as plastic bodies.
Thus, the existence of structures with elasto-plastic properties in epithelial
sheets seems to be plausible. It seems reasonable to attribute these properties to
the main supporting membranes of the sheet - that is, to the surface and to the
basal membranes. The relative morphogenetic passivity of the surface membrane
in respect to the individual cells can be derived as well from Fig. 15B and the
corresponding comments.
These properties of epithelial membranes can be of morphogenetic significance
in preventing small and casual cell shifts (which are taking place from time to
time in the proximal regions of Hydrozoa colonies) and in stabilizing the results
of massive and vast shifts.
PE3IOME
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MOp(f)oreHeTHHecKHe KjieTOHHbie flBimeHHJi y Obelia loveni, O. geniculata H Dynamena
pumila.
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OKOJIO 15/*. y 0. loveni aHajiorHHHbie pocTOBbie nyjibcauHH HMeioT ne5-8 MHH H aMiuiHTyny ,11,0 5/*. PHTM pocTOBbix nyjibcauHH He coBna,o,aeT c
PHTMOM nepHOAHnecKHx coKpameHHH npoKCHMaubHOM MacTH u,eHOcapKa.
3. OnMcaHbi AHCTajibHo HanpaBJieHHbie ABHJKCHHJI OTAejibHbix KJieTOK B 3KT0,n,epMe
pacTymnx CTeGneii H 3anaTK0B nmpaHTOB. IToKa3aH 3HaHHTejibHbiH pa36poc B
CKOpOCTHXflBHHCeHHJISKTOAepMaJIbHbTX KJieTOK.
Morphogenesis of hydroidpolypes
337
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BAKER,
(Manuscript received 16 June 1971)